TY - JOUR A1 - Mitic, Kristina A1 - Grafe, Marianne A1 - Batsios, Petros A1 - Meyer, Irene T1 - Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum JF - Cells N2 - Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis. KW - nuclear pore complex KW - nucleoporins KW - semi-closed mitosis KW - centrosome KW - Dictyostelium Y1 - 2021 U6 - https://doi.org/10.3390/cells11030407 SN - 2073-4409 VL - 11 IS - 3 PB - MDPI CY - Basel ER - TY - JOUR A1 - Grafe, Marianne A1 - Hofmann, Phillip A1 - Batsios, Petros A1 - Meyer, Irene A1 - Gräf, Ralph T1 - In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH JF - Cells : open access journal N2 - We expressedDictyosteliumlamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-Delta NLS Delta CLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of theDictyosteliumlamin, they are likely relevant also for wild-type lamin. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina Y1 - 2020 U6 - https://doi.org/10.3390/cells9081834 SN - 2073-4409 VL - 9 IS - 8 PB - MDPI CY - Basel ER - TY - JOUR A1 - Grafe, Marianne A1 - Hofmann, Phillip A1 - Batsios, Petros A1 - Meyer, Irene A1 - Gräf, Ralph T1 - In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH JF - Cells N2 - We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-∆NLS∆CLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina Y1 - 2020 VL - 9 IS - 8 PB - MDPI CY - Basel ER - TY - JOUR A1 - Grafe, Marianne A1 - Batsios, Petros A1 - Meyer, Irene A1 - Lisin, Daria A1 - Baumann, Otto A1 - Goldberg, Martin W. A1 - Gräf, Ralph T1 - Supramolecular Structures of the Dictyostelium Lamin NE81 JF - Cells N2 - Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina KW - expansion microscopy Y1 - 2019 U6 - https://doi.org/10.3390/cells8020162 SN - 2073-4409 VL - 8 IS - 2 PB - Molecular Diversity Preservation International CY - Basel ER - TY - JOUR A1 - Gräf, Ralph A1 - Grafe, Marianne A1 - Meyer, Irene A1 - Mitic, Kristina A1 - Pitzen, Valentin T1 - The dictyostelium centrosome JF - Cells : open access journal N2 - The centrosome of Dictyostelium amoebae contains no centrioles and consists of a cylindrical layered core structure surrounded by a corona harboring microtubule-nucleating gamma-tubulin complexes. It is the major centrosomal model beyond animals and yeasts. Proteomics, protein interaction studies by BioID and superresolution microscopy methods led to considerable progress in our understanding of the composition, structure and function of this centrosome type. We discuss all currently known components of the Dictyostelium centrosome in comparison to other centrosomes of animals and yeasts. KW - microtubule-organizing center KW - microtubule-organization KW - centrosome KW - Dictyostelium KW - mitosis Y1 - 2021 U6 - https://doi.org/10.3390/cells10102657 SN - 2073-4409 VL - 10 IS - 10 PB - MDPI CY - Basel ER - TY - JOUR A1 - Szabo, Istvan A1 - Grafe, Marianne A1 - Kemper, Nicole A1 - Junker, Ernst A1 - Malorny, Burkhard T1 - Genetic basis for loss of immuno-reactive O-chain in Salmonella enterica serovar Enteritidis veterinary isolates JF - Veterinary microbiology N2 - Fifty-two rough Salmonella enterica serovar Enteritidis (S. Enteritidis) isolates from broilers and the environment were characterized for their serological and genotypic properties. Under routine diagnostic serotyping methods such isolates lack the immuno-reactivity of the O-chain of the lipopolysaccharide (LPS), and are referred to as non-typeable. Using a modified slide agglutination method, the isolates could be differentiated into three different serological variants. Twenty-six isolates (50%) were defined as semi-rough, nineteen isolates (37%) as deep-rough, four isolates (8%) as rough and three isolates could not be assigned. Genetically, all semi-rough isolates lacked the wzyB gene encoding the O-antigen polymerase. Two isolates carried a frameshift mutation in wzyB. In 15 of 23 cases deep-rough or rough isolates had a single point mutation, a single- or double-nucleotide insert or deletion in the wbaP gene. The mutational changes lead to expression of truncated (premature) protein, resulting in the loss of the immuno-reactive O-chain. Both rough and smooth S. Enteritidis isolates showed identical or highly similar XbaI-PFGE profiles. Our results indicate that the loss of a functional LPS in S. Enteritidis isolates is caused by a variety of different mutation events within the wzyB (semi-rough) or the wbaP (deep-rough) gene and is not a result of a vertical spread of a specific S. Enteritidis subtype. The defect of the LPS may be a common evolutionary mechanism through which host defence can be escaped. KW - Salmonella KW - Enteritidis KW - Rough KW - Molecular typing KW - DNA sequencing Y1 - 2017 U6 - https://doi.org/10.1016/j.vetmic.2017.03.033 SN - 0378-1135 SN - 1873-2542 VL - 204 SP - 165 EP - 173 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Schweigel, Ulrike A1 - Batsios, Petros A1 - Müller-Taubenberger, Annette A1 - Gräf, Ralph A1 - Grafe, Marianne T1 - Dictyostelium spastin is involved in nuclear envelope dynamics during semi-closed mitosis JF - Nucleus N2 - Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae. KW - Spastin KW - LEM-domain KW - ESCRT KW - sun1 KW - dictyostelium KW - nuclear envelope KW - mitosis Y1 - 2022 U6 - https://doi.org/10.1080/19491034.2022.2047289 SN - 1949-1034 SN - 1949-1042 VL - 13 IS - 1 SP - 144 EP - 154 PB - Taylor & Francis Group CY - Philadelphia ER -